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Post-embryonic neurogenesis relies on the presence of neural stem cells, which are characterized by their multipotency and unique ability to self-renew. Despite their importance for the homeostasis and repair of the central nervous system, the transcriptional network governing stemness in adult neural stem cells is largely unknown. We established the transcription factor Rx2 as proxy for retinal stem cells in the post-embryonic retina of the teleost medaka (O. latipes). By interrogating the regulatory input to the Rx2 cis-regulatory element, we identified four transcription factors (Sox2, Tlx, Gli3, Her9), which distinctly shape the stem cell domain and modulate stem cell features in the retina. First of all, we analyzed the gene expression and found that these genes have distinct spatio-temporal expression patterns in the retinal stem cell domain. Conditional mosaic analysis in vivo confirmed Sox2 and Tlx as activators of Rx2. The ectopic expression of Sox2 or Tlx was sufficient to trigger de-differentiation of post-mitotic neurons and induced stem cell features therein. Conversely, sustained ectopic expression of Gli3 or Her9 repressed Rx2. Gain of Gli3 or Her9 in retinal stem cells arrested cell cycle progression and proliferation. Modification of conserved binding sites in the Rx2 cis-regulatory element revealed the importance of Sox and Gli transcription factors for the precise spatial Rx2 expression in retinal stem cells. We propose that the combinatorial regulatory input of Sox2, Tlx, Gli3, Her9 confines Rx2 expression and other features of retinal stem cells specifically to the periphery of the stem cell domain in the post-embryonic retina. To elucidate the functional role of Rx2 itself, mutants were established with the aid of targetable nucleases. Transcription activator-like effector nucleases were employed to induce double-strand breaks specifically in the Rx2 coding sequence, which in the case of erroneous non-homologous end-joining created sequence alterations at the site of cleavage. The generation of stable, heritable mutations in the endogenous Rx2 locus described here opens the opportunity for future genetic studies of Rx2 in medaka.
|Supervisor:||Wittbrodt, Prof. Dr. Joachim|
|Date of thesis defense:||13 March 2014|
|Date Deposited:||25 Mar 2014 09:18|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Service facilities > Centre for Organismal Studies Heidelberg (COS)
|Subjects:||000 Generalities, Science|